Assessment of stromal tumor infiltrating lymphocytes and immunohistochemical features in invasive micropapillary breast carcinoma with long-term outcomes.

PURPOSE: We studied the long-term outcomes of invasive micropapillary carcinoma (IMPCs) of the breast in relation to stromal tumor infiltrating lymphocytes (sTILs), prognostic biomarkers and clinicopathological features. METHODS: Stage I-III IMPCs treated with upfront surgery at our institution (January 2000 and December 2016) were included. Central pathology review was performed and sTILs (including zonal distribution and hot spot analysis) and tumor-associated plasma cells (TAPC) were evaluated. Expression of P53, BCL2, FOXP3, and WT1, which are variably linked to breast cancer prognosis, was measured by immunohistochemistry using tissue microarrays. Time-to-event endpoints were distant recurrence free interval (DRFI) and breast cancer-specific survival (BCSS). RESULTS: We included 111 patients of whom 59% were pure IMPCs. Standard clinicopathological features were comparable between pure and non-pure IMPCs. Overall, the mean sTILs level was 20% with higher proportion of sTILs present at the invasive front. There were no significant differences between pure- and non-pure IMPCs in sTILs levels, nor in the spatial distribution of the hot spot regions or in the distribution of TAPC. Higher sTILs correlated with worse DRFI (HR = 1.55; p = 0.0172) and BCSS (HR = 2.10; p < 0.001). CONCLUSIONS: Clinicopathological features, geographical distribution of sTILs and TAPC are similar between pure and non-pure IMPCs. Despite a high proportion of grade 3 tumors and lymph node involvement, we observed a low rate of distant recurrences and breast cancer-related death in this cohort of stage I-III IMPCs treated with primary surgery. Caution in interpretation of the observed prognostic correlations is required given the very low number of events, warranting validation in other cohorts.

A comparison of a brachytherapy and an external beam radiotherapy boost in breast-conserving therapy for breast cancer: local and any recurrences.

PURPOSE: Adding a tumour bed boost to whole-breast irradiation in breast-conserving therapy reduces local recurrence rates. The purpose of the present study was to investigate whether the boost technique influences the magnitude of the effect. METHODS: Patients treated with breast-conserving therapy for invasive breast cancer between 2000 and 2007 were included in the analysis. Three groups were considered according to the applied boost technique: electrons, brachytherapy or photons. The endpoints were local recurrence and any recurrence. Cox regression models were used and correction for the confounders in the association between boost technique and outcome was performed using multivariable models. RESULTS: 1879 tumours were included in the analysis. 1448 tumours (77.1%) were treated with an electron boost, 334 (17.8%) with a brachytherapy boost and 97 (5.2%) with a photon boost. Median follow-up was 13.1 years. The 10-year local recurrence rate was 2.2%. In multivariable analysis with correction for age, pathological Tumour or Node stage (pT, pN), chemotherapy and hormonal therapy, there was no significant difference between the three groups for the local recurrence risk (p = 0.89). 10-year any recurrence rate was 10.8%. In multivariable analysis with correction for age, pT, pN, resection margins, radiotherapy, year of diagnosis, chemotherapy and hormonal therapy, there was no significant difference between the brachytherapy group and the electron group or the photon group (p = 0.11 and p = 0.28, respectively). The photon group had more recurrences compared to the electron group (Hazard Ratio 1.81, 95% Confidence Interval 1.12; 2.92, p = 0.02). CONCLUSIONS: The local recurrence risk reduction of the tumour bed boost in breast-conserving therapy is not influenced by the applied boost technique.

Validation of a normal tissue complication probability model for late unfavourable aesthetic outcome after breast-conserving therapy.

PURPOSE: To validate a normal tissue complication probability (NTCP) model for late unfavourable aesthetic outcome (AO) after breast-conserving therapy. MATERIALS/METHODS: The BCCT.core software evaluated the AO using standardized photographs of patients treated at the University Hospitals Leuven between April 2015 and April 2016. Dose maps in 2 Gy equivalents were calculated assuming α/ß = 3.6 Gy. The discriminating ability of the model was described by the AUC of the receiver operating characteristic curve. A 95% confidence interval (CI) of AUC was calculated using 10,000 bootstrap replications. Calibration was evaluated with the calibration plot and Nagelkerke R2. Patients with unfavourable AO at baseline were excluded. Patient, tumour and treatment characteristics were compared between the development and the validation cohort. The prognostic value of the characteristics in the validation cohort was further evaluated in univariable and multivariable analysis. RESULTS: Out of 175 included patients, 166 were evaluated two years after RT and 44 (26.51%) had unfavourable AO. AUC was 0.66 (95% CI 0.56; 0.76). Calibration was moderate with small overestimations at higher risk. When applying all of the univariable significant clinicopathological and dosimetrical variables from the validation cohort in a multivariable model, the presence of a seroma and V45 were selected as significant risk factors for unfavourable AO (Odds Ratio 4.40 (95% CI 1.96; 9.86) and 1.14 (95% CI 1.03; 1.27), p-value <.001 and .01, respectively). CONCLUSIONS: The NTCP model for unfavourable AO shows a moderate discrimination and calibration in the present prospective validation cohort with a small overestimation in the high risk patients.

Validation of a prognostic scoring system for postmastectomy locoregional recurrence in breast cancer.

BACKGROUND: To date, it remains unclear which patients with breast cancer (BC) benefit from post-mastectomy radiotherapy (PMRT). Cheng et al. developed and validated a scoring system based on 4 prognostic factors for locoregional recurrence (LRR) to identify patients in need for PMRT. These factors include age, estrogen receptor status, lymphovascular status and number of affected axillary lymph nodes. PURPOSE: To validate the scoring system for LRR in BC developed by Cheng et al. by using an independent BC database. METHODS AND MATERIALS: We retrospectively identified 1989 BC cases, treated with mastectomy (ME) with or without PMRT at the University Hospitals Leuven between 2000 and 2007. The primary endpoint was 5-year locoregional control rate with and without PMRT, according to the LRR score. RESULTS: Median follow-up time was 11.4 years. After excluding patients with missing variables 1103 patients were classified using the LRR scoring system: 688 (62.38%) patients were at low risk of recurrence (LRR score 0-1), 335 (30.37%) patients were at intermediate risk of recurrence (LRR score 2-3) and 80 (7.25%) patients were at high risk of recurrence (LRR score ≥4). 5-year locoregional control rates with and without PMRT were 99.20% versus 99.21% (p = 0.43) in the low-risk group; 98.24% versus 85.74% (p < 0.0001) in the intermediate-risk group and 96.87% versus 85.71% (p = 0.10) in the high-risk group respectively. CONCLUSION: Our validation of the LRR scoring system suggests it can be used to point out patients that would benefit from PMRT. We recommend further validation of this scoring system by other independent institutions before application in clinical practice.

Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status.

PURPOSE: To determine the role of DNA repair in hypoxic radioresistance. METHODS AND MATERIALS: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. RESULTS: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficient line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. CONCLUSION: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity.

Fusion of ETV6 to GOT1 in a case with myelodysplastic syndrome and t(10;12)(q24;p13).

The ETV6 gene on 12p13 is involved in different hematologic malignancies through a variety of chromosomal translocations. Here we report the analysis of a t(10;12)(q24;p13) identified in a patient with myelodysplastic syndrome. Our results show that the t(10;12) results in the generation of an in-frame fusion between ETV6 and GOT1, a gene encoding a cytosolic glutamic-oxaloacetic transaminase enzyme. In addition, two other fusion transcripts involving exons from yet unidentified genes were detected, as well as expression of an uncharacterized gene from the 10q24 region. This work identifies GOT1 as a novel fusion partner of ETV6 in myelodysplastic syndrome.

Recurrent rearrangement of the Ewing's sarcoma gene, EWSR1, or its homologue, TAF15, with the transcription factor CIZ/NMP4 in acute leukemia.

Fusions of the TET-proteins (TLS/FUS, EWSR1, and TAF15/RBP56) to different transcription factors are involved in various malignancies including Ewing's sarcoma, primitive neuroectodermal tumors, and acute myeloid leukemia. These are thought to arise through transcriptional deregulation, with the transcription factor defining the tumor phenotype. We show that, as result of a t(12;17)(p13;q11) or its variant t(12;22)(p13;q12), the transcription factor gene CIZ/NMP4 is recurrently involved in acute leukemia through fusion with either EWSR1 or TAF15. The fusions possess transforming properties in NIH3T3 cells but do not affect the expression of CIZ target genes, suggesting a contribution to oncogenesis that is independent of the transactivating properties of the fusion protein. These results also extend the involvement of TET-protein fusions to acute lymphoblastic leukemia and suggest a role for CIZ/NMP4 in lymphoid and myeloid development.

Long term experience with 3D image guided brachytherapy and clinical outcome in cervical cancer patients.

BACKGROUND AND PURPOSE: To report our 10years' experience and learning curve of the treatment of cervical cancer patients with chemo radiotherapy and MRI (or CT in 9 selected patients) guided brachytherapy using pulsed dose rate (PDR) brachytherapy (BT). METHODS AND MATERIALS: Hundred and seventy consecutive patients with cervical cancer FIGO stage IB-IVB (without metastases beyond the para-aortic nodal region) were treated in our institute between 2002 and 2012. Patients received external beam radiotherapy (nodal boost to the lymph nodes positive at diagnosis)±chemotherapy followed by a pulsed or low dose rate brachytherapy boost. MRI (or CT) images were taken with the applicator in situ. The first 16 patients were treated according to X-ray-based plans, optimized on MRI. High-risk CTV, intermediate-risk CTV, bladder, rectum and sigmoid were retrospectively contoured according to the GEC-ESTRO recommendations. In all other patients, treatment plans were optimized after delineation of the target volumes and organs at risk at MRI (or CT). Doses were converted to the equivalent dose in 2Gy (EQD2) by applying the linear quadratic model. The median age of the patients was 55years (range 16-88). 41% had stage III or IV disease. Of the 170 patients, 91 patients had on imaging metastatic lymph nodes at diagnosis (62 patients pelvic lymph node involvement and 29 para-aortic). In 27 (16%) patients the intracavitary technique was combined with interstitial brachytherapy. RESULTS: The mean D90 and D100 for the high-risk CTV were 84.8±8.36Gy and 67.5±6.29Gy for the entire patient group. Mean D90 and D100 values for the IR CTV were 68.7±5.5Gy and 56.5±6.25Gy. There was an important learning curve between both patient groups, with an increase in mean D90 of 75.8Gy for the first 16 patients compared to 85.8Gy for the second group. At the same time, the mean dose to 2cm3 of bladder and sigmoid decreased from 86.1Gy to 82.7Gy and from 70Gy to 61.7Gy, respectively. At a median follow-up of 37months (range 2-136months), local control rate for all patients was 96%, the regional control (pelvic and para-aortic) rate 81% and crude disease free survival rate 55%. The overall survival at 5years is 65%. The higher dose to the target volume resulted in an increase in local control from 88% in the first 16 patients compared to 97% in the second patient group. Regarding late toxicity, 21 patients (12%) presented grade 3-4 late morbidity. Rectal, urinary, sigmoid and vaginal morbidity was 5%, 6%, 2% and 5%, respectively. A correlation between rectal D2cm3 >65Gy and grade >3 late morbidity was found (p=0.006). CONCLUSION: Although the majority of the patients presented with locally advanced carcinoma, excellent local and regional control rates were achieved. Rectal, urinary, sigmoid and vaginal grade 3-4 morbidity was 5%, 6%, 2% and 5%, respectively. A correlation between rectal D2cm3 >65Gy and grade >3 late morbidity was found (p=0.006).

Validation of the Web-Based IBTR! 2.0 Nomogram to Predict for Ipsilateral Breast Tumor Recurrence After Breast-Conserving Therapy.

PURPOSE: To evaluate the IBTR! 2.0 nomogram, which predicts 10-year ipsilateral breast tumor recurrence (IBTR) after breast-conserving therapy with and without radiation therapy for breast cancer, by using a large, external, and independent cancer center database. METHODS AND MATERIALS: We retrospectively identified 1898 breast cancer cases, treated with breast-conserving therapy and radiation therapy at the University Hospital Leuven from 2000 to 2007, with requisite data for the nomogram variables. Clinicopathologic factors were assessed. Two definitions of IBTR were considered where simultaneous regional or distant recurrence were either censored (conform IBTR! 2.0) or included as event. Validity of the prediction algorithm was tested in terms of discrimination and calibration. Discrimination was assessed by the concordance probability estimate and Harrell's concordance index. The mean predicted and observed 10-year estimates were compared for the entire cohort and for 4 risk groups predefined by nomogram-predicted IBTR risks, and a calibration plot was drawn. RESULTS: Median follow-up was 10.9 years. The 10-year IBTR rates were 1.3% and 2.1%, according to the 2 definitions of IBTR. The validation cohort differed from the development cohort with respect to the administration of hormonal therapy, surgical section margins, lymphovascular invasion, and tumor size. In univariable analysis, younger age (P=.002) and a positive nodal status (P=.048) were significantly associated with IBTR, with a trend for the omission of hormonal therapy (P=.061). The concordance probability estimate and concordance index varied between 0.57 and 0.67 for the 2 definitions of IBTR. In all 4 risk groups the model overestimated the IBTR risk. In particular, between the lowest-risk groups a limited differentiation was suggested by the calibration plot. CONCLUSIONS: The IBTR! 2.0 predictive model for IBTR in breast cancer patients shows substandard discriminative ability, with an overestimation of the risk in all subgroups.

Evaluation of a breast cancer nomogram to predict ipsilateral breast relapse after breast-conserving therapy.

BACKGROUND AND PURPOSE: A nomogram to predict for the 10-year ipsilateral breast relapse (IBR) after breast-conserving therapy (BCT) for breast cancer (BC) was developed based on the 'boost-no-boost'-trial with a concordance probability estimate (CPE) of 0.68. The aim of our study was to validate that algorithm. MATERIAL AND METHODS: We retrospectively identified 1787 BC cases, treated with BCT and radiotherapy at the University Hospitals Leuven from 2000 to 2007, without missing data of the nomogram variables. Clinicopathologic factors were assessed. Validity of the prediction model was tested in terms of discrimination and calibration. RESULTS: Median follow-up time was 10.75years. The validation cohort differed with respect to the administration of a radiation boost, chemo- or hormonal therapy, age, tumour diameter or grade, ductal carcinoma in situ and hormone receptor positivity. On multivariable analysis, the omission of the boost was a significant prognosticator of IBR (p<0.01). The 10-year IBR-rate was 1.4%. The nomogram demonstrated suboptimal discrimination (CPE 0.54) and calibration, with an overestimation of the IBR-risk in general. CONCLUSIONS: The predictive model for IBR in BC is imperfect in this more recent study population.

Is the use of a preoperative computed tomography beneficial to reduce the interobserver variability of the CTVboost delineation for breast radiation therapy?

PURPOSE: To determine whether the use of a preoperative (preop) computed tomography (CT) reduces (1) the clinical target volume boost (CTVboost) and (2) the interobserver variability (IOV) of the delineated CTVboost in breast radiation therapy. METHODS AND MATERIALS: In patients treated with breast-conserving therapy, 3 CT scans in treatment position were performed: (1) preop; (2) after surgery, prechemotherapy (postop); and (3) postchemotherapy (postchemo). Six radiation-oncologists delineated the tumor bed and CTVboost before and after fusion of the preop CT. To assess the IOV, the Jaccard index was used. Linear mixed models were performedfor all analyses. RESULTS: Eighty-two lumpectomy cavities were evaluated in 22 patients. No difference in CTVboost using the fusion of the preop CT (50.0 cm3; 95% confidence interval [CI], 35.6-64.4) compared with no fusion (49.0 cm3; 95% CI, 34.6-63.4) (P = .6) was observed. A significant increase in IOV was shown with the fusion of the preop CT; the mean Jaccard index of the CTVboost delineation of postop and postchemo CT together without the fusion of the preop CT was 0.53 (95% CI, 0.49-0.57) versus 0.50 (95% CI, 0.46-0.53) with fusion (P < .0001). CONCLUSIONS: There is no benefit of using a preop CT to reduce the volume or the interobserver variability of the delineated CTVboost for breast radiation therapy.

Boost delineation in breast radiation therapy: Isotropic versus anisotropic margin expansion.

PURPOSE: The purpose of this article is to compare isotropic and anisotropic margin expansion with regard to the size of the clinical target volume boost (CTVboost) and the interobserver variability (IOV). METHODS AND MATERIALS: Lumpectomy cavities marked with 3 or more surgical clips were delineated by 6 radiation oncologists who specialized in breast radiation therapy. CTVboost anisotropic was created by manually expanding the tumor bed with an anisotropic margin of 15 mm (20 mm in case of extensive intraductal component) minus the surgical free margins in 6 directions (anteroposterior, craniocaudal, and superoinferior). For the CTVboost isotropic, the tumor bed was enlarged with an isotropic margin of 15 mm (20 mm in case of extensive intraductal component) minus the minimal surgical free margin. The volumes of the delineated CTVboost (cm3) were measured. To assess the IOV, the Jaccard index (JI), defined as the intersection divided by the size of the union of the sample sets, was used (ideal value = 1). The JI was calculated for each case and each observer pair. Linear mixed models were used for all analyses. RESULTS: A total of 444 delineated tumor beds were evaluated. The mean volume of the CTVboost almost doubled by expanding the tumor bed with an isotropic margin compared with anisotropic margins (CTVboost isotropic 94 mL [12.5-331.0] vs CTVboost anisotropic 50 mL [3.2-332.7]; P = .0006). The IOV, assessed by the JI, significantly decreased by using isotropic versus anisotropic margin expansion (JICTV boost isotropic 0.73 [0.02-0.92] vs JICTV boost anisotropic 0.51 [0.0-0.8]; P< .0001). Because of the known positive correlation of the IOV and larger volumes, we corrected for CTVboost volumes. With this correction, the difference in IOV remains highly significant (P < .0001) in favor of isotropic margin expansion. CONCLUSIONS: The use of anisotropic margin expansion from tumorbed to CTVboost isotropic significantly reduced the volume of the delineated CTVboost with a factor of 1.9 compared with isotropic margin expansion, but it substantially increased the interobserver variability.

Image guided brachytherapy in locally advanced cervical cancer: Improved pelvic control and survival in RetroEMBRACE, a multicenter cohort study.

PURPOSE: Image guided brachytherapy (IGBT) for locally advanced cervical cancer allows dose escalation to the high-risk clinical target volume (HRCTV) while sparing organs at risk (OAR). This is the first comprehensive report on clinical outcome in a large multi-institutional cohort. PATIENTS AND METHODS: From twelve centres 731 patients, treated with definitive EBRT±concurrent chemotherapy followed by IGBT, were analysed. Kaplan-Meier estimates at 3/5years were calculated for local control (LC, primary endpoint), pelvic control (PC), overall survival (OS), cancer specific survival (CSS). In 610 patients, G3-4 late toxicity (CTCAEv3.0) was reported. RESULTS: Median follow up was 43months, percent of patients per FIGO stage IA/IB/IIA 22.8%, IIB 50.4%, IIIA-IVB 26.8%. 84.8% had squamous cell carcinomas; 40.5% lymph node involvement. Mean EBRT dose was 46±2.5Gy; 77.4% received concurrent chemotherapy. Mean D90 HRCTV was 87±15Gy (EQD210), mean D2cc was: bladder 81±22Gy, rectum 64±9Gy, sigmoid 66±10Gy and bowel 64±9Gy (all EQD23). The 3/5-year actuarial LC, PC, CSS, OS were 91%/89%, 87%/84%, 79%/73%, 74%/65%. Actuarial LC at 3/5years for IB, IIB, IIIB was 98%/98%, 93%/91%, 79%/75%. Actuarial PC at 3/5years for IB, IIB, IIIB was 96%/96%, 89%/87%, 73%/67%. Actuarial 5-year G3-G5 morbidity was 5%, 7%, 5% for bladder, gastrointestinal tract, vagina. CONCLUSION: IGBT combined with radio-chemotherapy leads to excellent LC (91%), PC (87%), OS (74%), CSS (79%) with limited severe morbidity.

Thymidine analogues to assess microperfusion in human tumors.

PURPOSE: To validate the use of the thymidine analogues as local perfusion markers in human tumors (no labeling indicates no perfusion) by comparison with the well-characterized perfusion marker Hoechst 33342. METHODS AND MATERIALS: Human tumor xenografts from gliomas and head-and-neck cancers were injected with iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) and the fluorescent dye Hoechst 33342. In frozen sections, each blood vessel was scored for the presence of IdUrd/BrdUrd labeling and Hoechst in surrounding cells. The percentage of analogue-negative vessels was compared with the fraction of Hoechst-negative vessels. Collocalization of the two markers was also scored. RESULTS: We found considerable intertumor variation in the fraction of perfused vessels, measured by analogue labeling, both in the human tumor xenografts and in a series of tumor biopsies from head-and-neck cancer patients. There was a significant correlation between the Hoechst-negative and IdUrd/BrdUrd-negative vessels in the xenografts (r = 85, p = 0.0004), despite some mismatches on a per-vessel basis. CONCLUSIONS: Thymidine analogues can be successfully used to rank tumors according to their fraction of perfused vessels. Whether this fraction correlates with the extent of acute hypoxia needs further confirmation.

A comparison of three different radiotherapy boost techniques after breast conserving therapy for breast cancer.

PURPOSE: Compare different boost techniques after breast conserving therapy (BCT) in terms of local and loco-regional recurrences. MATERIALS AND METHODS: From 2000 to 2005, patients treated with BCT for invasive breast cancer (BC) were included. An electron boost (EB) was performed for a superficial boost-volume (less than 29 mm under the epidermis), in all other cases a brachytherapy boost (BTB) was proposed. When patients refused a BTB or it was not possible for technical reasons, a photon boost (PB) was given. The primary endpoints were local and loco-regional recurrences. Secondary endpoints were metastasis-free and overall survival. RESULTS: 1379 patients were eligible for analysis. Most patients (1052) received an EB, 225 a BTB and 76 a PB. At a median follow-up of 8.8 years, 35 (2.5%) patients developed a local or loco-regional recurrence. Ten years local relapse-free rate was 97.9%. No differences between boost techniques were observed in relapse risk, metastasis-free and overall survival after multivariate analyses. CONCLUSION: In women treated with BCT followed by a boost irradiation to the tumor bed, no difference in local and loco-regional recurrence, metastasis-free and overall survival was observed comparing three different boost techniques. Outcome was excellent regardless of the boost technique.

HIF-1A, pimonidazole, and iododeoxyuridine to estimate hypoxia and perfusion in human head-and-neck tumors.

PURPOSE: Tumor hypoxia measured by microelectrodes has been shown to indicate poor patient outcome. Here we investigated four potentially more widely applicable immunohistochemical parameters of tumor oxygenation and perfusion in human head-and-neck tumors. METHODS: Twenty patients with squamous cell carcinomas of the head and neck treated with primary surgery were injected with pimonidazole and IdUrd the evening before operation. Consecutive paraffin-embedded sections were stained for blood vessels, pimonidazole, IdUrd, and HIF-1alpha. IdUrd labeling and Ki-67 labeling around individual blood vessels were scored. The spatial relationship between HIF-1alpha and pimonidazole was studied, as well as the distribution of both markers as a function of distance from the nearest blood vessel. RESULTS: Measurement of all four parameters (diffusion-limited fraction, pimonidazole fraction, HIF-1alpha fraction, IdUrd-negative vessels) was feasible, and a significant difference between tumors was found for all parameters. IdUrd-labeled cells were absent around some vessels, indicating lack of perfusion, because these regions were positive for Ki-67. There was a positive correlation between diffusion-limited fraction and pimonidazole area for all images from all tumors, although no correlation for mean values per tumor. Colocalization of pimonidazole and HIF-1alpha was low (0.02%-25%). Most expression profiles showed a more homogenous distribution for HIF-1alpha than pimonidazole. There was no significant correlation between the pimonidazole and HIF-1alpha fractions in the 10 tumors studied. CONCLUSIONS: Simultaneous immunohistochemical measurements related to hypoxia and perfusion are feasible (and easily applicable) in resected human tumors. The different geographic distributions of HIF-1alpha and pimonidazole indicate that HIF-1alpha might not be suitable as a marker for chronic hypoxia. Each parameter will be correlated with outcome in a larger ongoing study on head-and-neck tumors treated with surgery with or without postoperative radiotherapy.

Differentiation-associated staining with anti-pimonidazole antibodies in head and neck tumors.

BACKGROUND AND PURPOSE: Hypoxia is a strong negative prognostic factor for all three major treatment modalities for cancer. The bioreductive drug pimonidazole is currently under clinical investigation as a hypoxia marker. In human head and neck tumors, in addition to staining patterns typical of chronic hypoxia, staining was seen specifically around areas of keratinization, raising the question of whether this is hypoxia-related. This could influence quantitative hypoxia estimates using this marker. We investigated here whether the differentiation-related staining was caused by locally high reductive enzyme levels. PATIENTS AND METHODS: The nitrotetrazolium compound NBT was used, which is reduced by nitroreductases to yield a blue color. The assay was validated on three genetically related MDA231 human mammary carcinoma cell lines: wildtype, overexpressing DT-diaphorase (DT1), and overexpressing cytochrome p450 reductase (R4). Increased NBT staining under normoxia was indeed seen for both R4 and DT1 lines. Pimonidazole staining under normoxia was only seen in the R4 line. RESULTS: Frozen tumor sections from 20 patients with head and neck cancer injected with pimonidazole were incubated with NBT. Parallel sections were stained for pimonidazole. Staining patterns were then compared on matched images, and areas of keratinization scored for the presence or absence of pimonidazole and NBT. Pimonidazole staining was seen in 56% of keratinized areas, and of these, 78% showed increased NBT staining, indicating that high reductase levels are not a necessary requirement for differentiation-associated pimonidazole staining. In a second series, frozen sections of tumors from 15 patients not receiving pimonidazole were incubated with NBT and compared with staining after incubation with pimonidazole under both oxic and hypoxic conditions. Pimonidazole staining of some keratinizing areas under oxic conditions was seen. Of these areas, only a proportion (70%) showed increased NBT staining, confirming the lack of correspondence between keratin-associated pimonidazole staining and reductase levels. CONCLUSION: Hypoxia-independent pimonidazole staining can occur in more differentiated head and neck tumors, necessitating caution in hypoxia quantification. These data argue against a causative role for locally high reductase levels in differentiation-associated staining. DT-diaphorase appears to play no role in pimonidazole reduction.

Local relapse after breast-conserving therapy for ductal carcinoma in situ: a European single-center experience and external validation of the Memorial Sloan-Kettering Cancer Center DCIS nomogram.

PURPOSE: Adjuvant treatments after breast-conserving surgery (BCS) for ductal carcinoma in situ to prevent local relapse are considered standard of care. However, patient selection to prevent increased morbidity without proven survival benefit remains a challenge. To predict the risk of ipsilateral breast tumor relapse (IBTR) after BCS, the Memorial Sloan-Kettering Cancer Center (MSKCC) developed a nomogram. The aim of this study was to develop our own prediction model for IBTR and to provide an external validation of the MSKCC nomogram. METHODS: From 1973 to 2010, 467 patients were treated with BCS for ductal carcinoma in situ at the University Hospital Leuven. Clinicopathologic and treatment parameters of all patients were used to create a multivariable model. The predictive value of the model was evaluated using the concordance index (C-index) and concordance probability estimate (CPE). Multiple imputation was used to account for missing data to allow the MSKCC model to be tested on 467 patients. RESULTS: Median follow-up was 7.2 years, with 48 women who developed an IBTR. Omission of adjuvant endocrine therapy, younger age, and positive or close surgical margins were significantly associated with an increased risk of IBTR. The bootstrap-corrected C-index for 10-year prediction by our own model was 0.63 and the CPE was 0.61. The C-index and CPE for the 10-year relapse probabilities predicted by the MSKCC nomogram were 0.66 and 0.61, respectively. CONCLUSIONS: Despite the small number of events, the need for multiple imputation, and few patients without radiation, the MSKCC nomogram performance was somewhat better than our model. This shows that the MSKCC nomogram is externally valid. The MSKCC nomogram allows users to integrate the information from 10 different variables to provide a more precise risk stratification than the use of conventional single variables or hazard ratios.

Breathing adapted radiation therapy in comparison with prone position to reduce the doses to the heart, left anterior descending coronary artery, and contralateral breast in whole breast radiation therapy.

PURPOSE: To compare 3 different treatment positions in whole breast radiation therapy in terms of target volume coverage and doses to the organs at risk (OAR). METHODS AND MATERIALS: Thirty-four breast cancer (BC) patients (17 right-sided and 17 left-sided) were included in this dosimetric planning study. They all underwent a computed tomography (CT) scan in standard supine position in free-breathing (FB), supine position with gating in deep inspiratory breath hold (DIBH)(G), and prone position (P). Three-dimensional treatment plans were made for all 3 CTs. Target coverage and OAR sparing were evaluated. RESULTS: Breast volumes varied between 209 and 2814 cm(3). The target coverage, expressed as the mean volume of the breast receiving at least 95% of the prescription dose, was similar for the 3 treatment positions. The mean lung dose and the volume of the lungs receiving >20 Gy were significantly lower in P (1.7 Gy; 2.3%) compared with G (3.4 Gy; 5.6%; P < .0001) and FB (4 Gy; 7.3%; P < .0001). The volume of the contralateral breast receiving >5 Gy was significantly lower in G (P = .001) or FB (P = .004) versus prone. The supine position with gating in DIBH significantly reduced the volume of the heart receiving >30 Gy (V30(heart)), the mean heart (D(heart)), and mean left anterior descending coronary artery (LAD) dose (D(LAD)) for left-sided BC patients (V30(heart) 0.9%, D(heart) 1.6 Gy, DLAD 22.4 Gy) with respect to FB (V30(heart) 4.3%, D(heart) 3.5 Gy, DLAD 30.9 Gy)(V30(heart) and mean D(heart): P ≤ .0001; mean D(LAD): P = .008) and P (V30(heart) 7.9%, D(heart) 5.4 Gy, D(LAD) 36.4 Gy)(V30(heart) and mean D(heart): P = .0004; mean D(LAD): P = .01). CONCLUSIONS: The coverage of the planning target volume breast was equal for the 3 treatment positions. The lowest doses to the lungs were achieved in prone. The heart, LAD, and contralateral breast were best spared in the supine position with gating in DIBH.

Interaction partners for human ZNF384/CIZ/NMP4--zyxin as a mediator for p130CAS signaling?

Transcription factor ZNF384/CIZ/NMP4 was first cloned in rat as a p130Cas-binding protein and has a role in bone metabolism and spermatogenesis. It is recurrently involved in translocations in acute lymphoblastic leukemia. Translocations t(12;17) and t(12;22) fuse ZNF384 to RNA-binding proteins TAF15 and EWSR1, while a translocation t(12;19) generates an E2A/ZNF384 fusion. We screened for ZNF384 interacting proteins using yeast two-hybrid technology. In contrast to its rat homolog, human ZNF384 does not interact with p130CAS. Zyxin, PCBP1, and vimentin, however, were identified as ZNF384-binding partners. Given the interaction between human zyxin and p130CAS, these results suggest that zyxin indirectly enables the interaction of ZNF384 with p130CAS which is described in rat.